Differential changes in mGlu2 and mGlu3 gene expression following pilocarpine-induced status epilepticus: A comparative real-time PCR analysis

Group II metabotropic glutamate (mGlu II) receptors subtype 2 and 3 (mGlu2 and mGlu3) are subtle regulators of neuronal excitability and synaptic plasticity in the hippocampus. In recent years, researchers have investigated the potential neuroprotective and anticonvulsant effects of compounds acting on mGlu II receptors. However, abnormal expression and function of mGlu2 and mGlu3 have been reported in temporal lobe epilepsy, a phenomena that may limit the therapeutic effectiveness of these potentially new antiepileptic drugs. Here, we investigated seizure-induced changes in mGlu2 and mGlu3 mRNA following pilocarpine-inducted status epilepticus (SE) and subsequent epileptogenesis. Relative changes in gene expression were assessed by comparative analysis of quantitative real-time PCR (qrtPCR) by the delta–delta CT method. Pilocarpine-treated and control rats were sacrificed at different periods (24 h, 10 days, one month and more than two months) following SE. Total RNA was isolated from microdissected dentate gyrus and processed for RT-PCR and qrtPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control gene. Analysis of relative quantification (RQ) ratios of mGlu2 and mGlu3 mRNA expression revealed a significant down-regulation of both targets at 24 h after SE. Gene expression partially recovered at 10 days following SE reaching control levels at one month after SE. Two month after SE, mGlu2 mRNA expression was significantly down-regulated to ~ 41% of control expression whereas mGlu3 mRNA was comparable to control levels. Our data indicate that mGlu2 and mGlu3 expression is dynamically down-regulated or selectively enhanced during critical periods of epileptogenesis. Seizure-induced differential dysregulation of mGlu2 and mGlu3 receptors may affect the availability of these molecular targets for therapeutic compounds in epilepsy

Deficit of Kcnma1 mRNA expression in the dentate gyrus of epileptic rats

Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel alpha-subunits after pilocarpine-induced status epilepticus. By using comparative real-time PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy

Deficit of Kcnma1 mRNA expression in the dentate gyrus of epileptic rats

Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel α-subunits after pilocarpine-induced status epilepticus. By using comparative realtime PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy

Structure of concanavalin A at pH 8: bound solvent and crystal contacts

9 pages, 3 figures, 4 tables.Concanavalin A has been crystallized in the presence of the ligand (6-S-[beta]-D-galactopyranosyl-6-thio)-cyclomaltoheptaose. The crystals are isomorphous to those reported for ConA complexed with peptides at low resolution (3.00-2.75 Å). The structure was solved at 1.9 Å, with free R and R values of 0.201 and 0.184, respectively. As expected, no molecules of the ligand were bound to the protein. Soaking in the cryobuffer left its fingerprint as 25 molecules of glycerol in the bound solvent, most of them at specific positions. The fact that a glycerol molecule is located in the sugar-binding pocket of each of the four subunits in the asymmetric unit and another is located in two of the peptide-binding sites suggests a recognition phenomenon rather than a displacement of water molecules by glycerol. Crystal contact analysis shows that a relation exists between the residues that form hydrogen bonds to other asymmetric units and the space group: contact Asp58-Ser62 is a universal feature of ConA crystals, while Ser66-His121, Asn69-Asn118 and Tyr100-His205 contacts are general features of the C2221 crystal form.We thank Professor GarcõÂa-Ruiz for his constant support and helpful discussions and suggestions, Dr Cartwright for reviewing the manuscript and Professor Kabsch for providing XDS free of charge.We acknowledge EMBO/DESY for beam time, Professor Martinez Ripoll for his kind support in the preliminary X-ray characterization using the rotating-anode generator at the Instituto de QuõÂmica Fisica Roca Solano (CSIC) and the referees for valuable comments.Work in AA's laboratory is funded by Spanish Plan Nacional grant BMC- 2002-04011-C03.Peer reviewe

“Upregulation of STREX splice variant of the large conductance Ca2+-activated potassium (BK) channel in a rat model of mesial temporal lobe epilepsy”

Functional properties of large conductance Ca2+ activated potassium (BK) channels are determined by complex alternative splicing of the Kcnma1 gene encoding the alpha pore-forming subunit. Inclusion of the STREX exon in a C-terminal splice site is dynamically regulated and confers enhanced Ca2+ sensitivity and channel inhibition via cAMP-dependent phosphorylation. Here, we describe a real time quantitative PCR (qPCR) approach to investigate relative changes in the expression of STREX and ZERO splice variants using a newly designed set of probes and primers for TaqMan-based qPCR analysis of cDNA from the rat dentate gyrus at different time points following pilocarpine-induced status epilepticus. Reduction in Kcnma1 gene expression is associated with a relative increase of STREX splice variant. Relative expression of STREX variant mRNA was increased at 10 days and at more than 1 month following status epilepticus. The biological consequences of seizure-related changes in alternative splicing of Kcnma1 deserve additional investigation

Downregulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures

Amyloid β peptides modify the expression of antioxidant repair enzymes and a potassium channel in the septohippocampal system

Alzheimer\u27s disease (AD) is a progressive, neurodegenerative brain disorder characterized by extracellular accumulations of amyloid β (Aβ) peptides, intracellular accumulation of abnormal proteins, and early loss of basal forebrain neurons. Recent studies have indicated that the conformation of Aβ is crucial for neuronal toxicity, with intermediate misfolded forms such as oligomers being more toxic than the final fibrillar forms. Our previous work shows that Aβ blocks the potassium (K(+)) currents IM and IA in septal neurons, increasing firing rates, diminishing rhythmicity and firing coherence. Evidence also suggests that oxidative stress (OS) plays a role in AD pathogenesis. Thus we wished to determine the effect of oligomeric and fibrillar forms of Aβ₁₋₄₂ on septohippocampal damage, oxidative damage, and dysfunction in AD. Oligomeric and fibrillar forms of Aβ₁₋₄₂ were injected into the CA1 region of the hippocampus in live rats. The rats were sacrificed 24 hours and 1 month after Aβ or sham injection to additionally evaluate the temporal effects. The expression levels of the K(+) voltage-gated channel, KQT-like subfamily, member 2 (KCNQ₂) and the OS-related genes superoxide dismutase 1, 8-oxoguanine DNA glycosylase, and monamine oxidase A, were analyzed in the hippocampus, medial, and lateral septum. Our results show that both forms of Aβ exhibit time-dependent differential modulation of OS and K(+) channel genes in the analyzed regions. Importantly, we demonstrate that Aβ injected into the hippocampus triggered changes in gene expression in anatomical regions distant from the injection site. Thus the Aβ effect was transmitted to anatomically separate sites, because of the functional coupling of the brain structures

Laura ++ : a Dalitz plot fitter

The Dalitz plot analysis technique has become an increasingly important method in heavy flavour physics. The Laura fitter has been developed as a flexible tool that can be used for Dalitz plot analyses in different experimental environments. Explicitly designed for three-body decays of heavy-flavoured mesons to spinless final state particles, it is optimised in order to describe all possible resonant or nonresonant contributions, and to accommodate possible violation effects

A Non-parametric Approach to Measuring the \kpi{} Amplitudes in \dpkkpi{} Decay

Using a large sample of \dpkkpi{} decays collected by the FOCUS photoproduction experiment at Fermilab, we present the first non-parametric analysis of the \kpi{} amplitudes in \dpkkpi{} decay. The technique is similar to the technique used for our non-parametric measurements of the \krzmndk{} form factors. Although these results are in rough agreement with those of E687, we observe a wider S-wave contribution for the \ksw{} contribution than the standard, PDG \cite{pdg} Breit-Wigner parameterization. We have some weaker evidence for the existence of a new, D-wave component at low values of the $K^- \pi^+$ mass.Comment: 13 pages 3 figure

A Study of D0 --> K0(S) K0(S) X Decay Channels

Using data from the FOCUS experiment (FNAL-E831), we report on the decay of $D^0$ mesons into final states containing more than one $K^0_S$. We present evidence for two Cabibbo favored decay modes, $D^0\to K^0_SK^0_S K^- \pi^+$ and $D^0\to K^0_SK^0_S K^+ \pi^-$, and measure their combined branching fraction relative to $D^0\to \bar{K} ^0\pi^+\pi^-$ to be $\frac{\Gamma(D^0\to K^0_SK^0_SK^{\pm}\pi^{\mp})}{\Gamma(D^0\to \bar{K} ^0\pi^+\pi^-)}$ = 0.0106 $\pm$ 0.0019 $\pm$ 0.0010. Further, we report new measurements of $\frac{\Gamma(D^0\to K^0_SK^0_SK^0_S)}{\Gamma(D^0\to \bar{K} ^0\pi^+\pi^-)}$ = 0.0179 $\pm$ 0.0027 $\pm$ 0.0026, $\frac{\Gamma(D^0\to K^0\bar{K} ^0)}{\Gamma(D^0\to \bar{K} ^0\pi^+\pi^-)}$ = 0.0144 $\pm$ 0.0032 $\pm$ 0.0016, and $\frac{\Gamma(D^0\to K^0_SK^0_S\pi^+\pi^-)}{\Gamma(D^0\to \bar{K} ^0\pi^+\pi^-)}$ = 0.0208 $\pm$ 0.0035 $\pm$ 0.0021 where the first error is statistical and the second is systematic.Comment: 11 pages, 3 figures, typos correcte